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So what exactly do I do? Let's start with the big picture...Benzene ring

Many chemicals that are released into the environment are extremely stable. This means that they hang around for a long time, and this is a major problem if they are damaging to health or the environment. One group of stable chemicals is the aromatic hydrocarbons. These contain a carbon ring structure that does not degrade easily.

Some bacteria make enzymes which can break these stable ring structures and convert them into useful metabolic compounds, and it is a group of these enzymes that I am studying. Enzymes are normally highly specific in the reactions they carry out, and it would be useful to redesign enzymes to break down a wider range of aromatic compounds. These might then be useful in preventing the release of dangerous, stable pollutants into the environment.

A lysozyme crystal

Before such enzymes can be designed, we need to learn more about how they catalyse the reactions they do. Working out the 3D structure of an enzyme is an important step in learning about how it functions. The classic way of doing this is to grow crystals of the enzyme. X-rays can then be fired through these crystals, and the pattern they make can be interpreted to give a structure. Once we know the 3D structure of the enzymes, we can try to work out where the active site is, and then which amino acids in the protein sequence are responsible for the specificity of the enzyme, and which physically take part in the catalysis. This can be done in a series of steps:

SDS-PAGE gel

1. Mutate specific points in the DNA sequence that codes for the enzyme;

2. Engineer bacteria to read the mutated DNA sequence and produce high yields of the modified enzyme;

3. Extract and purify the enzyme from the bacterial cells;

4. Carry out a series of different assays with the enzyme and analyse the results to see if the specific amino acid change has affected the binding or activity of the enzyme.

These are some of the techniques I am using in the lab as part of my PhD project.

Profile photograph 

Nathan Field

Nathan dot Field at warwick dot ac dot uk